Studies on nodulated soybean plants have indicated that the lack of molybdenum (Mo) can limit biological N.sub.2 fixation, as Mo supplementation can increase the rate of symbiotic N.sub.2 fixation (see references 4, 11, 14, 16 identified hereinafter). The abundance of Mo in the earth's crust is only at an average concentration of about 15 .mu.moles/Kg (22). Accordingly, it is unlikely that soil organisms such as legumes and their associated "root nodule" bacteria will commonly encounter high levels of Mo. Specific high affinity systems for Mo-sequestering would, therefore, be useful, especially in acidic soil environments where Mo is not readily available to the organism, and it would seem likely that N.sub.2 -fixing bacteria possess efficient mechanisms for scavenging trace levels of Mo.
High affinity molybdate sequestering processes have been described for N.sub.2 -fixing Klebsiella pneumoniae (12, 13, 25), Clostridium pasteruianum (5), and free-living (20), and bacteroid forms of B. japonicum (17). From these studies, it is clear that many N.sub.2 -fixing bacteria, even wild type Bradyrhizobium japonicum harbored in root nodules of soybean, have excellent Mo-sequestering abilities. Some nitrogenase systems are independent of the need for Mo (15), but it is not yet known if this is true for N.sub.2 -fixing systems of any of the rhizobia.
The metabolic processes between Mo sequestering and insertion into nitrogenase apparently involve several steps (10, 13, 25), including internalization from the free molybdate pool, and the synthesis of intermediates such as the iron molybdenum cofactor (25, 27). Studies thus far have implicated several Mo-binding intermediates prior to Mo bound in nitrogenase for several N.sub.2 -fixing bacteria (10, 21, 25) and at least 5 genes have been assigned roles in intracellular Mo metabolism in K. pneumoniae (13, 24-26). Most and perhaps all of these Mo-metabolizing genes are present in the symbiotic N.sub.2 -fixing bacterium B. japonicum as well (2, 26). For B. japonicum bacteroids, Mo is taken up by a high affinity process that is dependent on a transmembrane proton gradient (17). The Mo that is taken up by bacteroid suspensions in 1 min. is tightly associated with the cell, as most of it is not exchangeable with a 100-fold greater amount of exogenously-added Mo (17).
Mutants of B. japonicum in Mo metabolism and free-living N.sub.2 -fixation have previously been isolated (20). Some of the mutant strains appeared to be deficient in early Mo metabolism steps (such as binding Mo extracellularly) and other strains in later, presumably intracellular, Mo metabolism processes. Even though the phenotype of the mutants with respect to N.sub.2 -fixation was the primary interest, all of the mutants were isolated on the basis of the ability of added Mo to restore the activity of nitrate reductase, another Mo-containing enzyme.